Interaction of GCSF with the Endocannabinoid System in Promoting Brain Repair

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Background: G-CSF has been identified as a potential therapeutic agent for TBI. Administration of G- CSF soon after injury results in proliferation and release into the circulation of bone marrow-derived cells (BMDC). Monocytes from blood are recruited into the brain to the site of the lesion where they differentiate into microglia. These, in turn generate a number of neurotrophic factors and cytokines involved in repair and regenerative processes. Numerous studies indicate that TBI impacts the endogenous cannabinoid system (eCBs), altering expression of eCB receptors CB1 and CB2, and changing levels of the endocannabinoids anandamide (N-arachidonoyl-ethanolamine; AEA) and 2-AG (2- arachidonoylglycerol). Moreover, administration of cannabinoid agents have been shown to enhance recovery from TBI mediated by anti-inflammatory cytokines and regenerative processes that parallel those triggered by G-CSF. Specific Aims of this research program are designed to test the hypotheses a) that G-CSF interacts with the endocannabinoid system (eCS) to promote brain repair and b) that blockade of CB1 and/or CB2 receptors will diminish or enhance the brain’s repair response to TBI. Aim 1: To investigate the effects of G-CSF on the expression of CB1 and CB2 and their natural ligands in mouse brain (cortex, striatum and hippocampus) following TBI. Dependent variables: CB1 and CB2 receptor expression (mRNA and protein), levels of the eCB ligands AEA and 2-AG, extent of apoptosis, microgliosis, astrocytosis, neuro-inflammation, levels of neurotrophic factors (BDNF, GDNF); changes in hippocampal neurogenesis. Aim 2a: To determine if stimulation of the brain endocannabinoid system with a) an inhibitor of fatty acid amide (FAAH) to increase levels of AEA and 2AG, or b) administration of selective CB1 and CB2 receptor antagonists will potentiate (or diminish) the known beneficial effects of G- CSF on brain repair and recovery. Dependent variables are the same as in Aim 1, with the additional parameter of recovery of performance in the radial arm water maze (RAWM). Aim 2b: To determine the extent to which the CB1-R or the CB2-R is responsible for enhanced recovery from TBI, the CB1-R and CB2-R knockout mice will be studied using the protocol detailed in Aim1. Dependent variables: CB1 and CB2 receptor expression (mRNA and protein), levels of AEA, 2AG in cortex, striatum and hippocampus; extent of microgliosis and astrocytosis in these 3 brain regions; levels of BDNF and GDNF, hippocampal neurogenesis and recovery of performance in RAWM. Aim 3: To determine the role of the CB2 receptor in the mobilization of BMDC in mediating the beneficial effects of G-CSF, we will administer a CB2 antagonist (or utilize CB2-R knockout mice) to animals treated with G-CSF or vehicle. Tracking of BMDC will utilize chimeric mice that have had bone marrow transplants from transgenic “green mice”. Dependent variables: percentage of total circulating white blood cells that co-express GFP and markers of monocytes, quantitative estimates of the distribution GFP+ cells in the lesioned and contralateral hemisphere, proportion of GFP+ cells that differentiate into microglial phenotypes (M1, M2), profile of cytokines/chemokines, hippocampal neurogenesis, and performance in RAWM. Aim 4: Effects of delayed treatment (1, 2, 4 wks after “severe” TBI) with G-CSF on behavioral recovery and repair mediated by eCBs. Dependent variables: are the same as in Aim 1, with the additional parameter of rate of recovery of performance in RAWM. Impact on the Field of Brain Repair: The consequences of TBI, stroke and neurodegenerative diseases are highly prevalent in the veteran population. There is a need for effective, and safe therapies for these disorders. The optimal timing of G-CSF therapy after TBI, with or without cannabinoid agonists or antagonists as determined from this project, will be helpful in developing guidelines for treatment of humans who have suffered brain injury.